The proposed research will examine both mechanistic and topographical aspects of protein targeting to, translocation across, and integration into the membrane of the endoplasmic reticulum (ER). These processes will be observed from a unique perspective, that of the nascent polypeptide chain, by taking advantage of a class of Lys-tRNA analogs that were devised and developed by us. Fluorescent dyes or photoreactive moieties, covalently attached to a lysine side chain, will be incorporated into nascent chains in an in vitro translation system; nascent chains of defined lengths will be prepared by using secretory or membrane protein mRNAs truncated at specific locations. This novel approach creates fully- assembled translocation or integration intermediates that have a fluorescent or, photoreactive probe located at a specific site in a functional complex. During the past grant period, fluorescent probes in such intermediates demonstrated, directly, that secretory proteins pass through an aqueous pore that spans the entire bilayer, are sealed off from the cytoplasm by a tight ribosome-membrane junction, and are initially sealed off from the ER lumen by some sort of gate. In this application, vesicles reconstituted with various purified ER membrane proteins will be examined using the same techniques to identify which is (are) involved in forming the pore, the junction, and the gate. The inside diameter of the pore will be estimated from the size of molecules that cannot pass through it, while the outer diameter will be estimated using fluorescence energy transfer between a dye in the nascent chain and dyes at the phospholipid surface. Polarity-sensitive fluorescent probes in the middle of a transmembrane segment of a nascent membrane protein will reveal at what point the segment moves from the aqueous pore into the hydrophobic bilayer, and also whether this movement is altered in multiply-spanning proteins. This will be correlated with the time the segment leaves the translocon by determining when a segment containing a photoreactive probe ceases to react covalently with translocon components. The sensitivity of the ribosome-membrane junction to large cytoplasmic domains in membrane proteins and to multiple insertion events will be assessed using cytoplasmic quenching agents. These events will also be examined in reconstituted vesicles in order to identify the role played by various ER proteins during the selection, orientation, and insertion of transmembrane segments. The role of GTP in regulating, the SRP-mediated targeting of ribosomes to the ER membrane will be examined by quantifying the nucleotide dependence of SRP binding to ribosomes and signal sequences, while nucleotide affinities for SRP54 and SRP will be determined directly and at equilibrium using fluorescent-labeled GTP in the presence or absence of signal peptides.